Podcasts about Polymerase

Polymerase chain reaction (PCR) was discovered in 1983 by Kary Mullis and Michael Smith, who were jointly awarded the Nobel Prize in Chemistry in 1993. Since then, PCR has been a cornerstone method that has been a pillar of discovery and applied science. The various types of PCR are sometimes confusing, and the relative pros and cons of each method are not always clear, which is why it's so great to have this episode's guest explain them all in a simple and clear-cut way. Dave Bauer, PhD, is an Application Scientist at Thermo Fisher Scientific that specializes in real time PCR (qPCR) and digital PCR (dPCR).  He has an educational background in physics, mathematics, and biology, but what's more important is that Dave loves to help others learn and to break down a topic's complexities to make it more understandable and approachable.  In this episode we hear Dave explain the difference between qPCR and dPCR, the importance of Poisson statistics to dPCR, dead volume, reaction chamber volume consistency, and more.  We learn how qPCR and dPCR complement each other and how they relate to sequencing methods for applications like single nucleotide polymorphism (SNP) detection.  As you've come to expect from Absolute Gene-ius, you also get a good sense of who Dave is and how he got to his current role. We learn about how he knew right away that academia wasn't for him, how he ended up unexpectedly working in forensics after his PhD, and how he eventually landed in his current Application Scientist role. Dave shares some great insights and advice, including how students should care less about their degree's name and more about what techniques they're learning and using in their studies. Visit the Absolute Gene-ius page to learn more about the guest, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System.This episode includes the following sound effects from freesound.org, licensed under CC BY-ND 4.0:“Sax Jazz,” by alonart“Balloon Pop / Christmas cracker / Confetti Cannon,” by Breviceps“Crowd Cheering,” by SoundsExciting

I read from DNA polymerase to do.     The word of the episode is "DNR".     Theme music from Jonah Kraut https://jonahkraut.bandcamp.com/     Merchandising! https://www.teepublic.com/user/spejampar     "The Dictionary - Letter A" on YouTube   "The Dictionary - Letter B" on YouTube   "The Dictionary - Letter C" on YouTube   "The Dictionary - Letter D" on YouTube     Featured in a Top 10 Dictionary Podcasts list! https://blog.feedspot.com/dictionary_podcasts/     Backwards Talking on YouTube: https://www.youtube.com/playlist?list=PLmIujMwEDbgZUexyR90jaTEEVmAYcCzuq     dictionarypod@gmail.com https://www.facebook.com/thedictionarypod/ https://twitter.com/dictionarypod https://www.instagram.com/dictionarypod/ https://www.patreon.com/spejampar https://www.tiktok.com/@spejampar 917-727-5757

Please go to academiccme.com/ovariancancer/ and complete the evaluation to receive your CE/CME Credit.

BUFFALO, NY- September 20, 2022 – A new research paper was published in Oncotarget's Volume 13 on September 14, 2022, entitled, “Targeting Krebs-cycle-deficient renal cell carcinoma with Poly ADP-ribose polymerase inhibitors and low-dose alkylating chemotherapy.” Loss-of-function mutations in genes encoding the Krebs cycle enzymes Fumarate Hydratase (FH) and Succinate Dehydrogenase (SDH) induce accumulation of fumarate and succinate, respectively, and predispose patients to hereditary cancer syndromes including the development of aggressive renal cell carcinoma (RCC). Fumarate and succinate competitively inhibit αKG-dependent dioxygenases, including Lysine-specific demethylase 4A/B (KDM4A/B), leading to suppression of the homologous recombination (HR) DNA repair pathway. Researchers Daiki Ueno, Juan C. Vasquez, Amrita Sule, Jiayu Liang, Jinny van Doorn, Ranjini Sundaram, Sam Friedman, Randy Caliliw, Shinji Ohtake, Xun Bao, Jing Li, Huihui Ye, Karla Boyd, Rong Rong Huang, Jack Dodson, Paul Boutros, Ranjit S. Bindra, and Brian Shuch from Yale University School of Medicine, West China Hospital/School of Medicine, Wayne State University, and University of California, Los Angeles have developed new syngeneic Fh1- and Sdhb-deficient murine models of RCC, which demonstrate the expected accumulation of fumarate and succinate, alterations in the transcriptomic and methylation profile, and an increase in unresolved DNA double-strand breaks (DSBs). “In this study, we sought to examine the activity of combined TMZ and PARPi in Krebs-cycle-deficient renal cancer models. Fh1 and Sdhb are the murine counter part of human FH and SDHB, respectively.” The efficacy of poly ADP-ribose polymerase inhibitors (PARPis) and temozolomide (TMZ), alone and in combination, was evaluated both in vitro and in vivo. Combination treatment with PARPi and TMZ results in marked in vitro cytotoxicity in Fh1- and Sdhb-deficient cells. In vivo, treatment with standard dosing of the PARP inhibitor BGB-290 and low-dose TMZ significantly inhibits tumor growth without a significant increase in toxicity. These findings provide the basis for a novel therapeutic strategy exploiting HR deficiency in FH and SDH-deficient RCC with combined PARP inhibition and low-dose alkylating chemotherapy. “Using newly developed Fh1 and Sdhb deficient syngeneic mouse models, we demonstrate that oncometabolite-induced HR defects can be leveraged with PARPi treatment to enhance sensitivity to low-dose TMZ in Krebs-cycle-deficient renal cancer.” DOI: https://doi.org/10.18632/oncotarget.28273 Correspondence to: Ranjit S. Bindra & Brian Shuch – Emails: Ranjit.Bindra@yale.edu & bshuch@mednet.ucla.edu Keywords: FH, SDHB, renal cell carcinoma, PARP inhibitor, temozolomide About Oncotarget: Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, visit Oncotarget.com and connect with us on social media: Twitter – https://twitter.com/Oncotarget Facebook – https://www.facebook.com/Oncotarget YouTube – www.youtube.com/c/OncotargetYouTube Instagram – https://www.instagram.com/oncotargetjrnl/ LinkedIn – https://www.linkedin.com/company/oncotarget/ Pinterest – https://www.pinterest.com/oncotarget/ LabTube – https://www.labtube.tv/channel/MTY5OA SoundCloud – https://soundcloud.com/oncotarget For media inquiries, please contact: media@impactjournals.com.

Dr. Tejas Patil looks at PCR testing for NSCLC.

2/11/2022 Additional resources OHA: Find a COVID-19 vaccine OHA: COVID-19 vaccine information for parents, guardians and people under 18 OHA: COVID-19 vaccine boosters and third doses OHA: COVID-19 testing CDC: COVID-19 Testing: What You Need to Know CDC: COVID-19 Self-Testing Medline.gov – What are PCR tests? Federal Trade Commission: Don't assume every COVID-19 testing site is legit Oregon Department of Justice: COVID-19 Sales, Scams and Fraud U.S. Department of Health and Human Services: COVID-19 Scams OHA Facebook  Safe+Strong website Ask a Black Doctor on The Numberz REACH web page and REACH Facebook Reminders about health, safety and cleaning guidance Q1: What kind of COVID-19 tests are out there at the moment? COVID-19 tests can detect either SARS-CoV-2, the virus that causes COVID-19, or antibodies that your body makes after getting COVID-19 or after getting vaccinated. Tests for SARS-CoV-2 tell you if you have an infection at the time of the test. This type of test is called a “viral” test because it looks for viral infection. Antigen or Nucleic Acid Amplification Tests (NAATs) are viral tests.  Tests for antibodies may tell you if you have had a past infection with the virus that causes COVID-19. Your body creates antibodies after getting infected with SARS-CoV-2 or after getting vaccinated against COVID-19. These tests are called “antibody” or “serology” tests.  The CDC does not currently recommend antibody testing to find out if you have COVID-19. Only viral tests are recommended to detect current COVID-19 infection. Q2: Is it possible to get a false positive on a viral test? NAATs, or molecular tests, are considered the most accurate form of COVID-19 testing because they detect genetic material from the virus that causes COVID-19. Polymerase chain reaction (PCR) tests are a type of NAAT. These tests are processed by certified laboratories, with most results available in 2 to 5 business days. Unlike many other tests, PCR tests can detect the virus in the earliest stages of infection. Other tests may miss early signs of disease because there aren't enough viruses in the sample, or your body hasn't had enough time to develop an antibody response. This means the test can detect the virus even before you show symptoms of COVID-19. Antigen testing is faster, but not as accurate. It detects proteins from the virus that causes COVID-19. This means the test is best done when you are showing symptoms of COVID-19 or have a lot of virus in your system. False negatives can happen if your body doesn't have enough of the virus for an antigen test to detect.  This is why the Centers for Disease Control and Prevention (CDC) considers NAATs, such as PCR tests, the gold standard for COVID-19 testing.  The CDC also recommends getting a NAAT if an antigen test provides negative results, but you have symptoms of COVID-19. But overall, positive viral tests are right more than 95% of the time. Q3: What are some of the aspects to consider in self testing for COVID-19? COVID-19 self-tests at home are one of many risk-reduction measures, along with vaccination, masking, and physical distancing, that protect you and others by reducing the chances of spreading SARS-CoV-2, the virus that causes COVID-19.  A positive self-test result means that the test detected the virus, and you must isolate so as to reduce the risk of spreading disease to someone else.  A negative self-test result means that the test did not detect the virus and you may not have an infection, but it does not entirely rule out infection. Repeating the test within a few days, with at least 24 hours between tests, will increase the confidence that you are not infected. The best time to test is 3 to 8 following an exposure and/or when you have symptoms. Q4: What are some things to look out for if I think I may be giving my information to a fake COVID testing site? Some warning signs that you can look out for are things such as: A provider asking about your nationality or immigration status. A site asking for your Social Security number. No notice of privacy practices provided, or no explanation for how your personal data will be used. A provider demanding to see your passport or driver's license when you have other documents that show your insurance status. Employees at the site who are not wearing full protective gear. Misspellings or unusual names in the URL for the website where the testing provider required you to sign up online. An unsolicited call or text from the testing site. If you receive one, do not provide any personal information until you have confirmed that source of the call or text is legitimate. Q5: Where can I go to find legitimate COVID testing sites in my local area? If you are looking for information on where to get tested for COVID-19, you can contact your primary health care provider or visit Oregon Health Authority's COVID testing web page at healthoregon.org/covid19testing. Q6: At what point should I take an at-home COVID test? People with symptoms can take a rapid antigen test immediately, experts said, but those who have had a known exposure to the virus should wait three to five days before doing so. Testing too soon, before the virus has had a chance to replicate, increases the odds of a false negative. Q7: What has happened if self-tested results show “Invalid” or “Error”? This means your specimen may not have been collected correctly, or the test may have malfunctioned. Invalid test results are rare but can occur. If the self-test shows an invalid result or a test error, the test did not work properly. If this happens, refer to the instructions for use in the package insert and contact the manufacturer for assistance. When in doubt about the whole process then seek professional help. Local vaccine events Date Time Location AddressSaturday, Feb. 12 | 11 a.m. to 4 p.m. | Rosewood Initiative | 16126 SE Stark StPortland, OR Tuesday, Feb. 15 | 2 to 8 p.m. | Lloyd Center | 1260 Lloyd CenterPortland, OR Thursday, Feb. 17 | 8 a.m. to 2 p.m. | Lloyd Center | 1260 Lloyd CenterPortland, OR You can find more vaccine events at multco.us/vaccineclinics. Document accessibility: For individuals with disabilities or individuals who speak a language other than English, OHA can provide information in alternate formats such as translations, large print, or braille. Contact the Health Information Center at 1-971-673-2411, 711 TTY or COVID19.LanguageAccess@dhsoha.state.or.us.

Molecular Testing PearlsIn Part 4 of our Heme Path series, we thoroughly examine the details of molecular testing and how it relates to hematologic and oncologic malignancies I. Molecular Testing BasicsA. Provides a means of assessing patient's genotypes, specifically at smaller changes in the genetic information B. How is it performed?1. Polymerase chain reaction (PCR)-based testing, which involves using a specific primer that is complementary to the area of interest on the patient's DNA2. PCR can allow for both amplification and quantification of gene of interest C. Can look for either single gene mutations (faster) or a panel of mutations (slower but more information) also known as NGSII. Clinical Utility of Molecular TestingA. Very useful in risk stratification based on the mutations noted (some mutations are unfavorable and some are favorable)B. Certain genetic mutations have drugs that are effective against them, therefore provides information about targeted therapeutic options C. In hematologic malignancies, can be used to also assess response to treatment1. You can determine minimal residual disease or MRD2. Can look for a gene mutation that was present in the original cancer clone and see if there is any amount of residual cancer left over on the order of 1 in a million cellsD. In solid cancers, used to determine presence of genetic changes that have prognostic and targeted treatment implications1. BRAF V600E mutation in melanoma → BRAF inhibitor pill treatment2. EGFR mutation in lung cancer → EGFR inhibitor pill treatmentIII. How is molecular testing different than FISH?A. Both require choosing probes and understanding what you are looking for before running the testB. FISH (discussed in part 3!) reports out of 200 cells and provides information about only larger kilobase sized genetic changes (translocations, inversions, deletions)C. Molecular testing analyzes a much larger number of cells and can detect changes at the single base pair level. Much more detailed and microscopic evaluation of genetic changesIV. Single Gene Molecular TestingA. Look for a specific gene mutation (i.e. EGFR for lung cancer, BRAF for melanoma, FLT3-ITD for AML)B. Pros: 1. Faster turnaround time 2. Has a higher resolution and effective for detecting MRDB. Cons:1. Only looks for one genetic mutation as opposed to a panel like in NGS2. Some diseases ideally require understanding of multiple mutations not just one for prognostication and treatment planningV. Next Generation Sequencing (NGS)A. Allows to sift through a larger part of the genome to identify a panel of mutations B. Panel of mutations chosen is based on the clinical context1. For example: NGS for acute myeloid leukemia is much different than NGS testing for lung cancer as each cancer has a much different genetic mutation profileC. Overview of technical aspects of running NGS1. Massively parallel sequencing meaning that many tiny primers are used and the areas that primers encode may be overlapping2. A computer takes all of the smaller pieces and puts them together to determine the correct sequenceD. Pros:1. Gives us an understanding of many different mutations present based on the panel chosen2. Again, this has both prognostic and predictive treatment implicationsE. Cons:1. May find mutations of undetermined significance meaning we currently do not understand how these mutations will affect prognosis and treatment decisions2. Very time consuming (~2-4 week turnaround time)3. CostlyReferences:1. https://jamanetwork.com/journals/jamaoncology/fullarticle/2734828 - Quick overview of NGS2. https://ashpublications.org/blood/article/125/26/3996/34323/Minimal-residual-disease-diagnostics-in-acute - Look at table 1 to see the difference in sensitivity for MRD testing3. https://www.oncotarget.com/article/27602/text/ - Emphasizes prognostic relevance of EGFR mutations in NSCLC4. https://www.nejm.org/doi/full/10.1056/NEJMoa1612674 - Phase 3 trial showed that targeted treatment for EGFR mutation in NSCLC was superior to chemotherapy5. https://www.nejm.org/doi/full/10.1056/nejmoa1614359 - Phase 3 trial showed that targeted treatment of FLT3 mutation in AML improved outcomesPlease visit our website (TheFellowOnCall.com) for more information Twitter: @TheFellowOnCallInstagram: @TheFellowOnCallListen in on: Apple Podcast, Spotify, and Google Podcast

説明書を読むのが難しい問題、実験プロトコルの記述、ゲームのキャラクターセレクトと実験テーマ選定の類似性について話しました。Show notes 赤城 ミニ四駆 東大出身某プロゲーマー midiprep PCR Nature Protocols ドラクエの始まり マリオの進む向き ポケモン赤緑 ドラゴンクエストV 波動拳/昇竜拳コマンド スマブラ スノボ理論　… ep75 にて話しております。 マゴさん … 2D神 藤村さん ザンギエフ エドモンド本田 今夜勝ちたい攻略 わからん殺し ストリートファイターIII 3rd Strike Q ユリアン ガー不 減数分裂 tadasuのDの仕事 画狂老人卍 … 葛飾北斎 ウメハラさん 学振 … 日本学術振興会 対空 冨田勲 Moog 冨田勝 E-Cell ハーバート・サイモン 冨田LR法 … GLR法 カーネギーメロン大学 高橋恒一 メッセージ 但し書き … 正直後半の論理的整合性はめちゃくちゃで、coelaはゲーム、tadasuは研究の話をしているのでさらにめちゃくちゃですが、お互いの間ではテレパシーによって話が通じています。順番をグチャグチャにはなしているので、論理的整合性が一見ないのですが、個人の中ではそれっぽく存在しているので、わかりにくければどこかでまたやりたいです。まぁ雰囲気を楽しむってことで！ Editorial notes 頭の中ではいろいろとつながっているのですが話してみるとかなりとっちらかっていますね。二人の間では話が通じています。途中から大雨で水が流れる音が入っています笑(tadasu) ザンギとホンダは概念的な存在として取り上げるつもりが、彼らに対する感情がちょっと漏れてしまいました。(coela)

DNA and RNA, nucleic acid processes, Polymerase chain reaction

DNA and RNA, nucleic acid processes, Polymerase chain reaction

What is interstitial Cystitis or IC? Answer: A chronic bladder syndrome that causes immense amounts of pain. In this episode, Dr. Purser talks about new urinary genetics testing for bacteria (PCR or Polymerase chain reaction testing), new pain options, and a diet that actually work for interstitial cystitis. Follow Dr. Purser! Info: http://www.DanPurserMD.com Supplements: https://www.PhysicianDesigned.com Books: https://www.amazon.com/-/e/B00BIKSGVQ Facebook: https://www.facebook.com/danpursermd Facebook: https://www.facebook.com/PhysicianDesigned Twitter: https://twitter.com/danpursermd YouTube: https://www.youtube.com/danpursermd Instagram: https://www.instagram.com/danpurser.md Pinterest: https://www.pinterest.com/danpursermd

In This Episode: Erin and Weer’d discuss the latest Supreme Court case involving gun rights, and yes it went 9-0 in our favor! Both the ATF ruling on receiver definition and David Chipman's confirmation hearing as ATF director has yet to materialize.  Is the Biden Administration even prioritizing gun control? A question was asked: How could Bill Maher could be fully vaccinated, yet test positive for COVID-19? Weer'd talks about what PCR is and how this might be possible. Xander gives us his Independent Thoughts on food prep and impulse buying. David talks about Pistol Caliber Carbines. Finally, Matt from Geeks, Gadgets and Guns gives us some survival tips for the open road. Did you know that we have a Patreon? Join now for the low, low cost of $4/month (that’s $1/podcast) and you’ll get to listen to our podcast on Friday instead of Mondays, as well as patron-only content like mag dump episodes, our hilarious blooper reels and film tracks. Show Notes Main Topic: Supreme Court rules warrantless home gun confiscation is unconstitutional in 9-0 vote Biden's Proposed Ghost Gun Rule Still Not Officially Published Bill Maher is Fully Vaccinated and Tested Positive for Covid Lab Alert: FDA Issues Safety Letter about Potential for False Positive Results with Antigen Tests for Rapid Detection of SARS-CoV-2 Polymerase chain reaction Gun Lovers and Other Strangers: Mauser C96 Beretta 93R Heckler & Koch VP70 CAA MCKS & Ronis Colt Model 635 Marlin Camp Carbine Ruger Deerfield Sig MPX Colt AR6951 Beretta Cx4 Storm Kel-Tec SUB2000 Hi-Point 995 Ruger PC Carbine CZ Scorpion Kriss Vector Tavor SAR  

On today’s ID the Future, physicist Brian Miller continues his conversation with host Eric Anderson. Here they explore more problems facing the idea that life began as strings of RNA. In their discussion of the RNA World Hypothesis and the origin of life generally, they touch on ideas advanced by Jeremy England, Jack Shostak, Nick Lane, Helen Hansma, and others. One of several big problems with the RNA-first hypothesis underscored by Miller and Anderson: For it to have even a slender chance of working, you need prebiotic Earth to generate not one but two information-rich RNA strands, and they somehow need to find each other before falling apart, and do so despite the fact that they aren’t looking for each Read More › Source

Today Dr Jay Richards joins us to discuss PCR testing and One Nation's Senator Malcolm Roberts says Australia Day is a celebration of our freedom. Dr Jay Richards, a Research Assistant Professor in the Busch School of Business at The Catholic University of America, and author of The Price of Panic? How The Tyranny of Experts Turned a Pandemic into a Catastrophe. The World Health Organization issued a notice cautioning testing facilities about interpretation of the Polymerase chain reaction tests or PCR tests and the risk of false positives. Do you see any significance in the notice or it's timing? The Pandemic has seen a total corruption of Government and bureaucracy globally. The media has become derelict in reporting the truth and are complicit with many Governments and the World Health Organization in the chaos that now prevails. Covid-19 has become Politics 101. Scare the hell out of the population, tell them that you will save them and then get elected. And as for the media, be incompetent and continue the narrative of the Government and keep the panic and fear as a headline and many many headlines daily. The UK Government is wanting to do the second jab, not at the 3 week interval, but extend it, untested to 12 weeks. Government leaders and public health officials have not provided credible justification for their draconian restrictions on businesses and individuals during the Covid-19 pandemic. In fact, looking at what's been happening in places such as California, New York and Washington, Government actions have more to do with politics than science. The lack of transparency and the narrow focus of media on Covid-19 infections has increased public fear and allowed governments the greatest latitude in destroying thousands of small businesses and lives, and taking away individual freedoms. The headline statistics on new infections in the US and in other countries have been alarming but do we expect to see a downturn in infections soon – and not just because of the vaccine. Tomorrow is Australia Day. Senator Malcolm Roberts discusses why we should be proud to be a part of this great country. The conversation around changing the date of Australia Day is part of the mainly leftist cancel culture movement, and they want it moved or totally abolished. Really for most Australians, Australia Day is our day to celebrate this great country. And most Australians celebrate it with a bbq and a beer or Aussie wine. It's a cross between America's July 4 Celebrations and thanksgiving. Check us out on twitter: @narrative2day Visit The Narrative at thenarrative.today

Basic Process Sample Collection Sample Transportation Nucleic Acid extraction and Purification Amplification and Detection Links https://en.wikipedia.org/wiki/Polymerase_chain_reaction https://en.wikipedia.org/wiki/Coronavirus https://www.k-state.edu/hermanlab/protocols/StandardPCRConditions.html https://en.wikipedia.org/wiki/RNA https://en.wikipedia.org/wiki/DNA https://en.wikipedia.org/wiki/Thermus_aquaticus

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.28.358481v1?rss=1 Authors: Kokic, G., Hillen, H. S., Tegunov, D., Dienemann, C., Seitz, F., Schmitzova, J., Farnung, L., Siewert, A., Hoebartner, C., Cramer, P. Abstract: Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3'-exonuclease that recognizes mismatches. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication. Copy rights belong to original authors. Visit the link for more info

はじめまして。九州大学ビジネススクールの荒木です。専門はバイオ産業です。北九州市戸畑区で生まれ、高校まで戸畑区にいました。大学と大学院でバイオインフォマティクスを勉強していました。その後、製薬会社やバイオベンチャー、海外の大学院への留学を経て、以前は大学の医学部や農学部で研究員として働いていました。 去年の３月から九州大学ビジネススクールでバイオ産業に関する講義を担当しています。バイオテクノロジーを使ってどういうビジネスが展開出来るかというようなお話をしています。薬から食品までバイオに関わる分野で幅広く活動してきました。 今日は今や国民の誰もが知っている「ＰＣＲ」は一体何なのか。どういう風に開発されていったのかについてお話しします。新型コロナウイルスの流行により、ＰＣＲ検査が有名になりました。 「ＰＣＲ」とは、"polymerase chain reaction"の略語です。 Polymeraseは遺伝子の素となるＤＮＡを合成する酵素。平たく言えばＤＮＡを作る装置のようなものです。chain reactionはそれを鎖みたいな形でどんどん繋げていって反応させる一連の反応のことです。 ＤＮＡを連続して合成していくというようなことがＰＣＲです。 今、皆さんが一番関心のあるコロナウイルスのＰＣＲ検査は実際何をやっているかと言うと、コロナウイルスに感染すると人間の体内にウイルスの遺伝子があるわけですが、少量ではなかなか人間が同定出来ないため、ＰＣＲによって人間が分かる量までウイルスの遺伝子を増やしています。逆に言えば、その遺伝子が増えるということは、コロナウイルスに感染しているということです。微量な遺伝子（ＤＮＡ）をどんどん増やしていく技術が「ＰＣＲ」です。「ＰＣＲ」によってウイルスが増えた人は陽性で増えなかった人は陰性という形で判定されます。 この技術は、ノーベル賞をとった技術です。ただ、非常に面白いことに、この技術は大学の研究者ではなくアメリカの一バイオベンチャーが開発し、1984年に実用化したものです。この開発の物語は非常に興味深く、後にこの技術を開発してノーベル賞をとったキャリー・マリス博士は、実はあまり優秀な科学者ではではありませんでした。開発当初は、そもそもこの会社の本丸的な研究テーマではなく、片手間にこの博士が一人でやっていたようです。この技術の原理自体は非常にシンプルで、実はＰＣＲ技術が開発される前の技術を組み合わせて成立していた技術です。ただ、そこに初めて気づいたのがこのマリス博士で、あまりにも簡単な原理だったため、本人もきっと他に誰かがやっていると思って過去の論文を色々見たけれども誰もやっていなかったため、更に開発を進めていきました。この会社はシータス社というバイオベンチャー企業ですが、そこのみんなは誰もこの技術の凄さに気付かず、見放されていたそうです。本当に何がどう凄くなるのかというのは誰もわからないという典型的な例だと思います。 その技術が今や世界で使われることになったわけです。残念ながらマリス博士は昨年亡くなられましたが、まさか自分が開発した技術が今これだけ世界中で使われているというのは彼も考えもしなかったのではないでしょうか。彼はシータス社とある意味喧嘩別れ的にこの会社を去っていくわけですが、その時の退職金はＰＣＲの発明を含めても１万ドルと言われています。後の経済効果を考えると非常に割安でした、その後ＰＣＲ技術は徐々にその凄さが世間に認知されるようになり、1993年にはノーベル化学賞を受賞することになります。それまでは大学の研究者がノーベル賞を受賞していたため、おそらく企業の研究者からノーベル賞が受賞された最初の事例でした。マリス博士は非常に変わり者で、大のサーフィン好きでノーベル賞の連絡があった時もサーフィンをしていたと言われています。結婚を4回されている非常に恋多き男でもあったようです。このＰＣＲは、日本車であるホンダのシビックでドライブ中に閃いたといわれています。 では、今日のまとめです。 現在世界中で使用されているＰＣＲは、キャリー・マリスという奇想天外な民間の科学者が閃いたアイデアによって開発されました。アイデアそのものは全く新しいものではなく、既存の組み合わせだったのですが、開発当時はその技術の凄さに誰も気付かず、認められるまでに数年かかりました。しかし、徐々にその技術の凄さが広がっていったということです。

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.20.347138v1?rss=1 Authors: Gong, S., Kirmizialtin, S., Chang, A., Mayfield, J. E., Zhang, Y., Johnson, K. A. Abstract: We examined the roles of Mg2+ ions in DNA polymerization by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase and by molecular dynamics simulation of Mg2+ binding. Binding of the Mg-nucleotide complex induces a conformational change of the enzyme from open to closed states in a process that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds weakly to the closed state of the enzyme-DNA-Mg.dNTP complex with an apparent Kd = 3.7 mM and facilitates the catalytic reaction. This weak binding of the catalytic Mg2+ is important to maintain fidelity in that the Mg2+ samples the correctly aligned substrate without perturbing the equilibrium at physiological Mg2+ concentrations. The binding of the catalytic Mg2+ increases nucleotide specificity (kcat/Km) by increasing the rate of the chemistry and decreasing the rate of enzyme opening allowing nucleotide release. Changing the free Mg2+ concentration from 0.25 to 10 mM increased nucleotide specificity (kcat/Km) by 12-fold. Mg2+ binds very weakly to the open state of the enzyme in the absence of nucleotide (Kd ~ 34 mM) and competes with Mg.dNTP. Analysis based on publish crystal structures showed that HIV RT binds only two metal ions during incorporation of a correct base-pair. MD simulations support the kinetic studies suggesting weak binding of the catalytic Mg2+ in open and closed states. They also support the two-metal ion mechanism, although the polymerase may bind a third metal ion in the presence of a mismatched nucleotide. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.18.303297v1?rss=1 Authors: Balint, E., Unk, I. Abstract: Polymerase eta (Pol{eta}) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Pol{eta} in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Pol{eta} proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here we show, that manganese triggers drastic changes in the activity of Pol{eta}. It increases the efficiency of ribonucleoside incorporation into RNA by ~400-2000-fold opposite undamaged DNA, and ~3000 and ~6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination almost lost during DNA synthesis by Pol{eta} with manganese. Moreover, Pol{eta} shows strong preference for manganese during RNA synthesis even at 25-fold excess magnesium concentration. Based on these, we suggest that selective metal cofactor usage plays an important role in determining the specificity of Pol{eta} during synthesis enabling it to function at both replication and transcription. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.12.248633v1?rss=1 Authors: Pauszek, R., Lamichhane, R., Rajkarnikar Singh, A., Millar, D. Abstract: Replication and repair of genomic DNA requires the action of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from E. coli, a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5' flap processing in separate domains. We show that a DNA substrate can spontaneously transfer between polymerase (pol) and 5' nuclease (5' nuc) domains during a single encounter with Pol I. Additionally, we show that the flexibly tethered 5' nuc domain adopts different positions within Pol I-DNA complexes, depending on the nature of the DNA substrate. Our results reveal the structural dynamics that underlie functional coordination in Pol I and are likely relevant to other multi-functional DNA polymerases. Copy rights belong to original authors. Visit the link for more info

COVID-19 Testing-Polymerase Chain Reaction (PCR) | Dr RR Baliga's 'Got Knowledge Doc' Podcasts for Physicians Not Medical Advice or Opinion

主播丨 丁教 嘉宾丨 廖国春 后期丨 迪卡普里鑫 ShowNotes丨 王雅 今天的节目依然为大家带来「疫情中的公司们」系列。我们希望能通过这个系列，分享创业者面对黑天鹅时的勇气，灵活，或许还有对这个世界的善意，并通过这些分享和大家一同面对这种不确定性。 疫情发生以来，试剂盒困局似乎始终没有完美的解决方案。除了使用试剂盒检测有很高专业门槛外，漏检率高也是无法忽视的一个问题。 这期节目我们采访了 IDbyDNA (https://www.idbydna.com)的创始人兼 CEO 廖国春，他为我们介绍了另一种正在蓬勃发展中的微生物感染检测技术：宏基因组检测。本期节目你将听到，为什么核酸检测盒的确诊率不尽如人意，在技术上是否有替代方案；医疗诊断问题在大数据的背景下如何转变为计算问题；更有价值的是，宏基因组检测可能为流行病的防控打开新的局面。 另外，硅谷早知道第四季已经上线，需要大家在喜马拉雅、苹果 Podcasts 等各大平台上重新订阅收听。那请大家接着关注声动活泼旗下其他节目。 本期主播 丁教，声动活泼联合创始人 本期嘉宾 廖国春， IDbyDNA 的创始人兼CEO The takeaway 宏基因组测序是把样品中所有生物的核酸拿过来不加区分的进行检测。测完之后实际上相当于把病人样品数据化了，这个时候就变成了一个计算问题，我们需要从样品上百万上千万或者更多的DNA序列片段里面找出来，哪些是从人来的，哪些是从微生物来的，哪些是有可能致病的。 传统检测方法PCR具有很高的特异性，需要设计一个高特异性的探针，所以它适用于目标明确且非常了解检测对象的情况。 核酸试剂盒（PCR）检测效率低有多方面的原因：在疫情早期我们对病毒的基因序列所知甚少，只能根据一些已公开发表的数据来设计探针，无法保证不漏检；当前生产压力大，人手不足，试剂盒的质检无法得到充分保证；上呼吸道和痰液并不是理想的病毒采样点，对病毒核酸的采样构成挑战。 宏基因组检测在流行病防控上的优势在于：第一，它可以同时检测很多病菌而不需要专门对某一种病菌采取特别措施，在大规模检测患者的时候可以在早期就发现问题。第二，测序完成后可以不仅可以判断是不是，还可以提供致病病菌的基因信息，有利于制定治疗方案，还能跟数据库里病菌进行比对，追溯传染源。 基因库的数据一方面来自于公共数据库，一方面来自于检测病例分析。 宏基因组测序想要保证质量，前期准备需要保证样品的制备技术和有效的内标，因为核酸是测序的基础，而内标可以判断实验流程是否完整；后期分析需要完备的数据库来对照分析，同时也要注意DNA序列片段的来自于保守的位点还是特异的位点，后者信息量高，更有分析价值。 本期讨论的主要问题 传统核酸检测技术在新冠检测中的局限性 新的技术如何解决这个问题 宏基因组检测的优势 宏基因组的商业模式和应用前景 宏基因组检测在流行病防控上的意义 访谈中提及名词 PCR：即聚合酶链式反应（Polymerase chain reaction），是一项利用DNA双链複製的原理，在生物体外複製特定DNA片段的核酸合成技术，具有较强的灵敏度，准确度和特异性。通过这一技术，可在短时间内大量扩增目的基因，而不必依赖大肠杆菌或酵母菌等生物体。此次新冠肺炎中用于确诊的试剂盒采用的就是逆转录PCR技术（RT-PCR），是聚合酶链式反应（PCR）的一种广泛应用的变形。在 RT-PCR 中，一条 RNA 链被逆转录成为互补 DNA，再以此为模板通过 PCR 进行 DNA 扩增。新型冠状病毒为单链RNA病毒，故采用该方法。 宏基因组：宏基因组（Metagenomics）又称元基因组，或称环境基因组，是指同时被研究的整个微生物群落的DNA。宏基因组测序，是对特定环境样品中的微生物群体的基因组（尤其是那些种类众多的难于培养的微生物），进行序列测定。宏基因组检测技术应用在病毒感染研究中，具有可以整体性观察，数据化样品等优点。 基因文库：将含有某种生物不同基因的许多DNA片段，导入受体菌的群体中储存，各个受体菌分别含有这种生物的不同的基因，称为基因文库。 DRG：即疾病诊断相关分组(Diagnosis Related Group)，是指以出院患者信息为依据，综合考虑患者的主要诊断和主要治疗方式，结合个体体征如年龄、 并发症和伴随病，将疾病的复杂程度和费用相似的病例分到同一个(DRG)组中， 从而让不同强度和复杂程度的医疗服务之间有了客观对比依据。在美国按照此制度，对DRGs中各类费用，均有一确定的费率，出院病人按其所属DRGs的费率向医院结帐，医院自负盈亏。这样，医院在提供医疗服务前就已知该组疾病资源消耗的最高限额，从而促使医院为获得利润主动降低经营成本，提高工作效率。也使医疗保险方对受保人每次住院费用都有准确的预算，便于控制费用。 23andMe：一家DNA鉴定公司，可以通过唾液检测DNA来得知你的血统混合情况，也可以选择提交你的DNA数据，用于遗传学研究。 LBT：全称Laboratory developed test，用来指代受CLIA监管的体外诊断。 FDA：即美国食品药品监督管理局（Food and Drug Administration），由美国国会即联邦政府授权，是专门从事食品与药品管理的最高执法机关， 寨卡病毒（Zika Virus）：一种通过蚊虫进行传播的虫媒病毒，非洲，美洲，亚洲以及太平洋均有发生疫情的记录。它会引发类似温和的形式的登革热的症状，目前还无法通过药物或疫苗来预防。 BGM If I Only - So Vea Eye of the Newscaster - Out To The World 微博：@声动活泼 微信公众号：@声动活泼 网站：shengfm.cn 支持/打赏我们：http://shengfm.cn/donation Special Guest: 廖国春.

Topic Discussed : Novel Technologies Driving Infectious Disease ManagementSpeaker: Deepak JayakumarKey Takeaways:Urgent need of rapid and accurate detection at the point of need is a prime driver of infection diagnosis. Frost & Sullivan’s research study explores some of the novel and emerging diagnostic technologies for infectious pathogen identification which includes: CRISPR-based paper diagnostic tests connected with a smartphone that can provide an accurate, rapid diagnosis with a minimum requirement of complex resourcesSome of the most recent development in antibody-antigen based assay and colorimetric assays are enlisted like saliva-based rapid diagnosis, 5-minute sepsis detection tests, improved immunoassay and mobile-based diagnostics for bacterial and Urinary Tract infectionUpgraded portable, fast, and next-generation Polymerase chain reaction technologies like ultra-fast PCR, dry reagents, sample to direct pathogen detection, portable real-time systems, multiplexed and improved PCR technologies are describedDiagnostics coupled with multidisciplinary approaches like artificial intelligence and machine learning in microscopy, sequencing; bioinformatics platforms and databases for pathogen identification, drug resistance and susceptibility testing which assist physicians to promptly start relevant treatments. Also, a fusion of one or more technologies such as applications of sound waves to develop diagnostic biosensors, applications of magnetic resonance in PCR and other technologies like nano-pore technology, microfluidics are discussedFor further insights, please join us for future podcasts and to know more about our Growth Partnership Services, reach out to digital@frost.com or click here to Contact Us.Related Keywords: Frost & Sullivan, Infectious disease diagnostics, Point of care, point of need, rapid diagnosis, PCR, antigen antibody assays, culturing, microscopy, microorganism identification, pathogen identification, CRISPR, next-generation sequencing, biosensors, nanopore, malaria, HIV, influenzaWebsite: www.frost.com See acast.com/privacy for privacy and opt-out information.

Steve and Caleb are back from Summer Break! The guys catch up on how they have been spending their summer. Steve has been busy working on his Jeep all summer. Steve talks about how much he loves working on cars as a hobby. The guys talk about how fun it would be to have an old project car to work on. They also fill us in on how they have been spending their time in the lab. Caleb has been working on figuring out third gen sequencing, which neither Steve nor anyone else has heard of. Caleb warns about the troubles of being on the cutting edge of science. Steve talks about how he has been working on doing qPCR for his project. The guys give a quick lesson on RNA and cDNA and why you would want to perform qPCR and they talk about some of the troubles of working with RNA. They also explain why funding random science is so important and how you never know what a project will lead to. Caleb explains the story of how Taq polymerase was discovered. Finally, it is unfortunate that the guys have to end on talking about the problem of gun violence. There was recently a shooting in the Oregon District in Dayton, Ohio, both Steve and Caleb's home town. The guys discuss how they feel about it and what a complicated problem it is. They also give a couple of suggestions of what small things you could do to possibly indirectly help with this issue.WebsiteInstagramFacebook

PCR法の発明者であるKary Mullis博士追悼回。PCRの原理と初期プロトコルやKary Mullisの生涯、爆速DNAポリメラーゼ、DNAを用いたハミルトン経路問題の計算（DNA computing）について話しました。Show notes Kary Mullis (Wikipedia)…PCRの発明者。1993年のノーベル化学賞を受賞 Kary Mullis (Web Site) Kary Mullis: Play! Experiment! Discover!…Kary MullisのTEDプレゼンテーション(2002)。 何か知りたいことがあれば、自分で実験し、観察し、記録し、法則性を見つけよ アイデアから始めよ、自分で試し、ドンドン改良せよ 権威に頼るな (時には必要であるが) 自分の行為に正直でなければならない Sons of Sputnik: Kary Mullis at TEDxOrangeCoast…青春時代、ロケットを作っていた話をKaryが熱心にしている。 Polymerase Chain Reaction: PCR (Wikipedia)…テンプレートDNAから目的領域のDNAを増幅させることができる。分子生物学に必須の基本方法。Denature->Annealing->Extensionのステップを繰り返すことでDNAは指数的に増幅されていく。例えば20回のサイクルではN^20=1,048,576分子に増える。 PCRのトラブルシューティングまとめ Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia…Saiki et al. Science 1985. PCRの最初の論文。現在とプロトコルはほとんど変わっていないが、当時はDNA polymeraseとして、Klenowを用いていたため、温度を上げてDenatureさせた後はKlenowを新しく入れる必要があった (高温になるとKlenowが失活するため)。このため、サイクル反応のたびにどんどん反応容量が増えていき、最終的には140uLのうち、30%ちかくがKlenowになる。 Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction…PCR初期の論文 Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction…PCR初期の論文 Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase…イエローストーン国立公園でとられた好熱細菌であるThermus aquaticusから取られたDNA polymeraseであるTaqを応用したPCRの論文。今のPCRの原型となる。被引用数としてはこの論文が一番PCR関連の中では多い。(2019年現在、被引用数は2万越え) Thermus aquaticus…イエローストーン国立公園で単離された好熱細菌。DNA polymeraseであるTaqはこの菌から取られた。 5x NEB Phusion Polymerase Buffer…このようにPCRのバッファーだけも売っている。 Thermus thermophilus…伊豆にある峰温泉で取られた好熱細菌。 数兆円の経済効果–PCRの発見…古市泰宏先生によるPCRをめぐる当時の状況とKary Mullisの回顧録。当時の雰囲気を感じ取ることができる。 Cosmological Significance of Time Reversal…Kary Mullisが24歳の時にかいた物理の論文。 KOD DNA Polymerase…国産の耐熱性DNAポリメラーゼ。鹿児島県小宝島の硫気孔から今中先生らにより単離された超好熱始原菌 Thermococcus kodakarensis KOD 1株に由来している。 小宝島…鹿児島の小宝島。一度聖地巡礼で行きたい。 小宝島小・中学校…小宝島には小中学校があり、生徒はいま10人、先生も10人だそうです。鹿児島の本港南埠頭から約12時間で行けるそうです。行ってみたい。のどかな雰囲気が伝わってくる。 超好熱菌由来の新規DNAポリメラーゼの発見とその産業利用…上記のKODの発見などに関する今中先生らによる記事。 峰温泉 PrimeSTAR® Max DNA Polymerase…Takaraの開発した伸長反応が5秒/1kbという驚異的な速度かつ精度を誇るDNAポリメラーゼ。 縁から中心を捉える科学－好熱菌を通して…大島泰郎先生による好熱菌研究のエピソード Leonard Adleman (Wikipedia)…DNA computingを初めて行ったAdlemanはRSA暗号の提案によって2002年にチューリング賞を受賞している。 Molecular computation of solutions to combinatorial problems. Science 1994…PCRを用いることによって7個の頂点からなるハミルトン経路問題を解くことを示した論文。DNA computingにおける最初の例。PDFが読めますので興味があればぜひ。 グラフ理論 (Wikipedia) RSA暗号 (Wikipedia)…RSAのAはAdlemanのA。 ハミルトン閉路問題…ハミルトン閉路問題における、閉路の条件を取り除いたものがハミルトン経路問題。 DNA computing (Wikipedia)…未踏の領域。 NP完全問題を解くDNAコンピューター…日本語のDNAコンピューターに関する解説 Solution of a 20-Variable 3-SAT Problem on a DNA Computer. Science 2002…AdlemanによるDNAコンピューター第二弾の論文。 Scaling up molecular pattern recognition with DNA-based winner-take-all neural networks. Nature 2018 Editorial notes DNAやその基礎反応を利用したコンピューティング技術についてもっと色々と考えていきたいですね (soh) アイデアを次々と試していきたい (tadasu)

From ASM Microbe 2019, Vincent, Brianne and Calvin meet up with Craig Cameron to discuss his career and his work exploring RNA-dependent RNA synthesis and single cell virology. Hosts: Vincent Racaniello, Brianne Barker, and Calvin Yeager Guest: Craig Cameron Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Cameron laboratory Curioscity podcast Ribavirin is an RNA virus mutagen (Nat Med) RNA virus error catastrophe: test using ribavirin (PNAS) Timestamps by Jolene. Thanks! Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

From ASM Microbe 2019, Vincent, Brianne and Calvin meet up with Craig Cameron to discuss his career and his work exploring RNA-dependent RNA synthesis and single cell virology. Hosts: Vincent Racaniello, Brianne Barker, and Calvin Yeager Guest: Craig Cameron Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Cameron laboratory Curioscity podcast Ribavirin is an RNA virus mutagen (Nat Med) RNA virus error catastrophe: test using ribavirin (PNAS) Timestamps by Jolene. Thanks! Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

A trio TWiVers reports on influenza in Australia, how a host protein impacts bird to human movement of influenza virus, and marine DNA viral diversity in the oceans from pole to pole. Hosts: Vincent Racaniello, Kathy Spindler, and Alan Dove Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Australian Influenza Surveillance Real-time tracking of influenza H3N2 ANP32B, or not to be (eLife) ANP32B on TWiV 377 Species specific differences in ANP32B for influenza virus replication (eLife) Marine viral DNA diversity pole to pole (Cell) Letters read on TWiV 554 Timestamps by Jolene. Thanks! Weekly Science Picks Alan - Stephanie Langel’s Discovery Talk Kathy- Map of languages in the USA Vincent- Grad student mental health with Susanna Harris video and on TWiM 199 Listener Picks Fernando - Pushing back on hyped-up science Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

A trio TWiVers reports on influenza in Australia, how a host protein impacts bird to human movement of influenza virus, and marine DNA viral diversity in the oceans from pole to pole. Hosts: Vincent Racaniello, Kathy Spindler, and Alan Dove Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Australian Influenza Surveillance Real-time tracking of influenza H3N2 ANP32B, or not to be (eLife) ANP32B on TWiV 377 Species specific differences in ANP32B for influenza virus replication (eLife) Marine viral DNA diversity pole to pole (Cell) Letters read on TWiV 554 Timestamps by Jolene. Thanks! Weekly Science Picks Alan - Stephanie Langel’s Discovery Talk Kathy- Map of languages in the USA Vincent- Grad student mental health with Susanna Harris video and on TWiM 199 Listener Picks Fernando - Pushing back on hyped-up science Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Team TWiV reveals DNA polymerases that do not require a primer, and packaging of hepatitis delta virus by the envelope glycoproteins of diverse viruses. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Brianne Barker Guest: Kiki Warren Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Bacteriophage DNA polymerase is primer-independent (PNAS) Primer-independent DNA polymerase from mobile elements (Cell Rep) Non-HBV helper viruses for HDV (Nat Comm) Waning of measles vaccine immunity (one, two, three) Letters read on TWiV 552 Timestamps by Jolene. Thanks! Weekly Science Picks Alan - Seek Rich - Want to See My Genes? Get a Warrant and NIH director will no longer speak on all-male science panels Brianne- Unique scientists Dickson- Vaccines - Calling the Shots Vincent- NY eliminates religious vaccine exemptions and Jessica Biel reveals that she does not understand vaccines Kiki- Converting type A to type O blood Listener Picks Justin - An Apple a Day Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Team TWiV reveals DNA polymerases that do not require a primer, and packaging of hepatitis delta virus by the envelope glycoproteins of diverse viruses. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Brianne Barker Guest: Kiki Warren Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Bacteriophage DNA polymerase is primer-independent (PNAS) Primer-independent DNA polymerase from mobile elements (Cell Rep) Non-HBV helper viruses for HDV (Nat Comm) Waning of measles vaccine immunity (one, two, three) Letters read on TWiV 552 Timestamps by Jolene. Thanks! Weekly Science Picks Alan - Seek Rich - Want to See My Genes? Get a Warrant and NIH director will no longer speak on all-male science panels Brianne- Unique scientists Dickson- Vaccines - Calling the Shots Vincent- NY eliminates religious vaccine exemptions and Jessica Biel reveals that she does not understand vaccines Kiki- Converting type A to type O blood Listener Picks Justin - An Apple a Day Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

In diesem Podcast meiner Schulreihe im Fach Biologie geht es um die Verdoppelung der DNA - die DNA Polymerase

In dieser Folge meiner Schulreihe geht es um die PCR - Methode, welche bei Kriminalfällen eingesetzt wird. Hier wird die DNA im Reagenzglas gezielt Vervielfältigt. Ich würde mich über ein LIKE und ein ABBO sehr freuen. Danke fürs zuhören und viel Spaß beim lernen! Instagram: adg.offiziell

The TWiV hosts discuss the distribution of prions in the eyes of patients with sporadic Creutzfeldt-Jacob disease, and the origins and evolution of RNA viruses. Hosts: Vincent Racaniello, Alan Dove, Rich Condit, Kathy Spindler Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Please take the TWiV listener survey ASV 2019 Satellite Symposia Crowdfunding for EV-D68 research Aaron Klug, 1926-2018 (Nature) Prions in sCJD eyes (mBio) Evolution of RNA viruses (mBio) Walter Gilbert's The RNA World (Nature) Image credit Letters read on TWiV 526 Timestamps by Jolene. Thanks! Weekly Science Picks Alan - Population mountains to visualize cities Rich- A Neanderthal Perspective on Human Origins with Svante Pääbo Kathy- NASA Space Mission posters Vincent - Package Thief vs Glitter Bomb Trap Listener Pick Neal- Was a Scientist Jailed? Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

The TWiV hosts discuss the distribution of prions in the eyes of patients with sporadic Creutzfeldt-Jacob disease, and the origins and evolution of RNA viruses. Hosts: Vincent Racaniello, Alan Dove, Rich Condit, Kathy Spindler Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Please take the TWiV listener survey ASV 2019 Satellite Symposia Crowdfunding for EV-D68 research Aaron Klug, 1926-2018 (Nature) Prions in sCJD eyes (mBio) Evolution of RNA viruses (mBio) Walter Gilbert's The RNA World (Nature) Image credit Letters read on TWiV 526 Timestamps by Jolene. Thanks! Weekly Science Picks Alan - Population mountains to visualize cities Rich- A Neanderthal Perspective on Human Origins with Svante Pääbo Kathy- NASA Space Mission posters Vincent - Package Thief vs Glitter Bomb Trap Listener Pick Neal- Was a Scientist Jailed? Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

In 1987 in Tunbridge Wells, just south of Dartford, two young women were murdered five months apart. Both lived alone in bedsits less than a mile from one another, both worked on Camden Road, both ate lunch at the same cafe. The women didn’t know each other, but they will forever be remembered together for their murderer was never caught. In 2007, with advances in forensics, a full DNA profile was obtained from minute evidence from one of the crime scenes. But would the killer be caught or is he still out there?Following each episode of Katherine Kelly's brand new TV series Murdertown, Benjamin Fitton from They Walk Among Us unpicks a new case from each location. Watch Murdertown exclusively on Crime+Investigation in the UK every Monday at 9pm from 3rd September and then straight after listen to a brand new story right here with the Murdertown podcast. You can get in touch using #Murdertown on Twitter, Facebook or Instagram.Crime+Investigation's Murdertown podcast is hosted by Benjamin Fitton, written by Anna Priestland, produced by Sam Pearson and Chloe Frost, and edited by James Collopy.SOURCES2007 APPEAL:http://news.bbc.co.uk/2/hi/uk_news/england/kent/8453919.stmhttp://news.bbc.co.uk/2/hi/uk_news/england/kent/6957446.stmhttp://news.bbc.co.uk/2/hi/uk_news/england/kent/6954760.stm25 YEAR ANNIVERSARY:https://www.mirror.co.uk/news/serial-killer-murdered-wendy-knell-905508https://www.heart.co.uk/kent/news/local/appeal-over-25-year-old-murder/http://www.bbc.com/news/uk-england-kent-18532610https://www.mirror.co.uk/news/serial-killer-murdered-wendy-knell-905508www.mirror.co.uk/news/serial-killer-murdered-wendy-knell-905508.ampBLOG:http://wolfiewiseguy.blogspot.com/2014/07/the-unsolved-murders-of-wendy-knell.htmlCRIMEWATCH:https://www.eveningtelegraph.co.uk/fp/crimewatch-coming-end-two-shocking-local-cases-appear-show-one-yet-solved/DNA:The Guardian: Case Closed 17th Jan 2008https://www.theguardian.com/uk/2008/jan/16/ukcrime.forensicsciencePCR:https://en.wikipedia.org/wiki/Polymerase_chain_reaction See acast.com/privacy for privacy and opt-out information.

At Tufts University Dental School in Boston, Vincent speaks with Katya Heldwein and Sean Whelan about their careers and their work on herpesvirus structure and replication of vesicular stomatitis virus. Host: Vincent Racaniello Guests: Katya Heldwein and Sean Whelan Become a patron of TWiV! Links for this episode Crystal structure of HSV gB (Science) Crystal structure of HSV fusion regulator gH-gL (Nat Struct Mol Biol) Nuclear Exodus: Herpesviruses Lead the Way (Ann Rev Virol) VSV Pseudotypes Bearing gB, gD, gH, and gL (J Virol) Recovery of infectious VSV from cDNA clones (PNAS) Structure of the L Protein of VSV (Cell) Unique strategy for mRNA cap methylation by VSV (PNAS) Molecular architecture of VSV RNA polymerase (PNAS) This episode is brought to you by the Defense Threat Reduction Agency. Part of the U.S. Department of Defense, the Agency’s Chemical and Biological Technologies Department hosts the 2017 Chemical and Biological Defense Science & Technology Conference to exchange information on the latest and most dynamic developments for countering chemical and biological weapons of mass destruction. Find out more at http://www.cbdstconference.com Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Brianne joins the TWiVMasters to explain how mutations in genes encoding RNA polymerase III predispose children to severe varicella, and detection of an RNA virus by a DNA sensor. Hosts: Vincent Racaniello and Rich Condit Guest: Brianne Barker Become a patron of TWiV! Links for this episode RNA pol III mutations underlie severe varicella (J Clin Inves) Dengue virus activates cGAS by release of mitochondrial DNA (Sci Rep) Dengue virus NS2B protein targets cGAS for degradation (Nat Micr) Image credit Letters read on TWiV 456 This episode is brought to you by the Defense Threat Reduction Agency. Part of the U.S. Department of Defense, the Agency’s Chemical and Biological Technologies Department hosts the 2017 Chemical and Biological Defense Science & Technology Conference to exchange information on the latest and most dynamic developments for countering chemical and biological weapons of mass destruction. Find out more at http://www.cbdstconference.com Weekly Science Picks Brianne - Up-Goer Five Challenge (related to Thing Explainer) Rich - The Farthest: Voyager in Space and Vipassana Momma: Science Vincent - Episode of Revisionist History: The Basement Tapes Listener Pick Jennie - What Piece of Lab Equipment Are You? Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Ben tenOever joins the TWiVoli to discuss the evolution of RNA interference and his lab's finding that RNAse III nucleases, needed for the maturation of cellular RNAs, are an ancient antiviral RNA recognition platform in all domains of life. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler Guest: Ben tenOever Become a patron of TWiV! Links for this episode RNAseIII is an ancient antiviral effector (Nature) Influenza suppression of RNAi (PNAS) Questioning mammalian antiviral RNAi (Nat Micro) Antiviral protection via RdRp (PLoS Path) Evolution of antiviral defense (Cell Host Micr) Letters read on TWiV 450 Weekly Science Picks Ben - Invisible Invaders by Peter Radetsky Kathy - ASV 2017 Virolympics Crossword solved (pdf) and National Museum of the Air Force Rich - Columbia river gorge and Undaunted Courage by Stephen Ambrose Dickson - All the World's Earthquakes Alan - Map of Roman roads Vincent - Brain atlas of fly behavior Listener Pick Rob - Vaccinate Your Kids and Charles Darwin Natural Selection Islam - Self-assembling virus and Virus Patterns Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Kat Arney explains how a protein that repairs broken DNA holds the key to killing cancer cells – but only if you stop it from doing its job

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler The TWiVniks review the past week's findings on Zika virus and microcephaly, and reveal a chicken protein that provides insight on the restriction of transmission of avian influenza viruses to humans.   Links for this episode Larvicide not involved in microcephaly (CBS News) Obama seeks $1.8b for Zika virus (Guardian) Zika virus in Puerto Rico (MMWR) Zika virus in semen (EID) Zika virus in Indonesia (EID) Phylogeny of Zika virus (EID) Zika virus may cause mental disease (NYTimes) Zika virus from amniotic fluid (Lancet) Host protein underlies avian influenza host restriction (Nature) Wendy Barclay on TWiV 177 Home to roost (Cell) Image credit Letters read on TWiV 377 This episode is sponsored by 32nd Clinical Virology Symposium and Microbe Magazine Podcast Weekly Science Picks Alan - CDC vaccine quizDickson - A telescope so powerfulVincent - Virology Lectures 2016 (virology blog, iTunes U)Rich - Ion propulsion (Wiki)Kathy - Spurious Correlations Listener Pick Johnye - Magellan telescope and van Egmond photograpy at Kids Should See This Send your virology questions and comments to twiv@microbe.tv

Kat Arney reveals how high temperature bacteria provide us with the molecule that speeds up DNA sequencing

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler The TWiVers explain how a protein platform assists the hepatitis C virus RNA polymerase to begin the task of making viral genomes. Links for this episode NYAS Afterschool STEM Mentoring Program Concerns about editing the human germline (Science, Nature) Engineering the perfect baby (MIT Tech Rev) RNA recognition by HCV polymerase (Science) Kickstarting an RNA polymerase (Science) A protein platform for priming (virology blog) Apoenzyme and holoenzyme (Wikibooks) Ebolavirus will not become a respiratory pathogen (virology blog) Burkholderia pseudomallei (CDC) Letters read on TWiV 330 Timestamps by Jennifer. Thank you! Weekly Science Picks Alan - X-Flare 2.1 capturedRich - Volvo ocean race drops research buoys (YouTube)Kathy - Nature double-blind peer reviewDickson - Largest cave in the worldVincent - New program for early-career scientists Listener Pick of the Week Michael - Space Engine and Kerbal Space ProgramVarun CN - Incognito by David Eagleman Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler The TWiVocateurs discuss how the RNA polymerase of enteroviruses binds a component of the splicing machinery and inhibits mRNA processing.   Links for this episode Viral polymerase binds Prp8 to inhibit mRNA splicing (PLoS Path) Prp8 protein of the spliceosome (RNA) Nuclear entry of poliovirus RdRp (Virol) RNA processing (TWiV 216) Measles in USA (CDC) Principles of Microbial Diversity Journal of Microbiology & Biology Education Letters read on TWiV 328 Weekly Science Picks Alan - Positively Negative (PLoS One)Rich - ISS assembly (YouTube)Kathy - Ancient treesDickson - Carolyn PorcoVincent - Tony Fauci on Ebola outbreak (YouTube) Listener Pick of the Week David - Shot by ShotJudi - The Power of Herd Immunity Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Hosts: Vincent Racaniello. Special guests: Laurene Mascola and David Persing Vincent meets up with Laurene and David at the Annual Meeting of the Southern California Branch of the American Society for Microbiology, where they discuss how the Los Angeles County Department of Health is preparing for an outbreak of Ebola virus infection, and Cepheid's game-changing, modular PCR system for the diagnosis of infectious diseases.

Hosts: Vincent Racaniello Guests: Laurene Mascola and David Persing Vincent meets up with Laurene and David at the Annual Meeting of the Southern California Branch of the American Society for Microbiology, where they discuss how the Los Angeles County Department of Health is preparing for an outbreak of Ebola virus infection, and Cepheid’s game-changing, modular PCR system for the diagnosis of infectious diseases.

Hosts: Vincent Racaniello. Special guests: Laurene Mascola and David Persing Vincent meets up with Laurene and David at the Annual Meeting of the Southern California Branch of the American Society for Microbiology, where they discuss how the Los Angeles County Department of Health is preparing for an outbreak of Ebola virus infection, and Cepheid’s game-changing, modular PCR system for the diagnosis of infectious diseases.

  Vincent meets up with Laurene and David at the Annual Meeting of the Southern California Branch of the American Society for Microbiology, where they discuss how the Los Angeles County Department of Health is preparing for an outbreak of Ebola virus infection, and Cepheid’s game-changing, modular PCR system for the diagnosis of infectious diseases.

Host: Vincent Racaniello Guests: Marshall Bloom, Sonja Best, and Byron Caughey Vincent visits the Rocky Mountain Laboratories in Hamilton, Montana and speaks with Marshall, Sonja, and Byron about their work on tick-born flaviviruses, innate immunity, and prion diseases. Links for this episode Rocky Mountain Laboratories, NIH Flavivirus persistence (Path Dis) Flaviviruses antagonize IRF1 (J Imm) Prion test using nasal brushings (NEJM) Video of this episode - view at YouTube Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

In the February 2014 issue of Clinical Chemistry, the influence of PCR reagents on DNA polymerase extension rates were studied by examining nucleotide incorporation with DNA dyes. We are joined by one of the authors of that study, Dr. Carl Wittwer. He is Professor of Pathology at the University of Utah Health Sciences Center and is also affiliated with ARUP Laboratories, BioFire Diagnostics, and is an Associate Editor of Clinical Chemistry.

Tjalle Hoekstra onderzocht DNA-replicatie door één enkel DNA-polymerasemolecuul tegelijk te observeren met behulp van een geavanceerde optische techniek, het optisch pincet. Hij ontdekte dat het verantwoordelijke eiwit DNA-polymerase veel vaker dan verwacht fouten corrigeert. Spreker: T.P. Hoekstra. Promotor: prof.dr.ir. G.J.L. Wuite, prof.dr.ir. E.J.G. Peterman Faculteit: Faculteit der Exacte Wetenschappen. Datum: 26-03-2014

Tjalle Hoekstra onderzocht DNA-replicatie door één enkel DNA-polymerasemolecuul tegelijk te observeren met behulp van een geavanceerde optische techniek, het optisch pincet. Hij ontdekte dat het verantwoordelijke eiwit DNA-polymerase veel vaker dan verwacht fouten corrigeert. Spreker: T.P. Hoekstra. Promotor: prof.dr.ir. G.J.L. Wuite, prof.dr.ir. E.J.G. Peterman Faculteit: Faculteit der Exacte Wetenschappen. Datum: 26-03-2014

Host: Vincent Racaniello Guests: W. Ian Lipkin and Thomas Briese Vincent meets up with Ian and Thomas to discuss their finding that MERS-coronavirus has been circulating in dromedary camels in the Kingdom of Saudi Arabia since at least 1992. Links for this episode MERS-CoV in dromedary camels (mBio) TWiV 251: Don't kiss the camel TWiV 247: Today's weather in virology TWiV 239: Filterable camels Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Thu, 16 Jan 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16826/

Dr. Stefan Zeuzem discusses his manuscript "Efficacy of the Protease Inhibitor BI 201335, Polymerase Inhibitor BI 207127, and Ribavirin in Patients With Chronic HCV Infection." To view the print version of this abstract go to: http://bit.ly/v0xg2a

Vincent, Rich, and Alan continue Virology 101 with a discussion of transcription, the process of making mRNA from a DNA template.

Form follows function heißt es in Design und Architektur. Doch nirgends folgt die Form der Funktion mehr als in der Natur. Strukturen sind es deshalb auch, die Professor Patrick Cramer, Direktor des Genzentrums der LMU, vor allem interessieren – um hoch komplexe Abläufe in der Zelle rekonstruieren zu können. Für die Entschlüsselung des Enzyms RNA-Polymerase II erhielt er mit dem Gottfried Wilhelm Leibniz-Preis 2006 die höchste wissenschaftliche Auszeichnung Deutschlands.

Cisplatin ist ein weit verbreitetes Zytostatikum, das seine Wirkung durch die Ausbildung von Quervernetzungen innerhalb eines DNA-Stranges entfaltet. Für das Cisplatin 1,2-d(GpG) Dinukleotid-Addukt konnte bereits sowohl in vitro als auch in Zellen die zentrale Bedeutung von DNA Polymerase η (Pol η) während des TLS-Prozesses nachgewiesen werden. In Zusammenarbeit mit der Gruppe Livneh wurde untersucht, ob der 1,3-d(GpTpG) Cisplatin Schaden ebenfalls in Zellen überlesen werden kann. Erstaunlicherweise sind humane Zellen in der Lage auch diesen komplexen Schaden zu überlesen, allerdings nur sehr ineffektiv. Da die Anzahl an Mutationen in Pol η defizienten Zellen deutlich erhöht war, konnte eine Beteiligung von Pol η am TLS-Prozess des 3’dGs des Schadens nachgewiesen werden. Somit konnte erneut die Initiator-Rolle von Pol η im Multi-Polymerasen Modell beim Überlesen von Cisplatin-Schäden bestätigt werden. Um mechanistische Details des Pol η vermittelten TLS-Prozesses zu erhalten, wurde die TLS-Fähigkeit von S. cerevisiae Pol η mittels in vitro Primerverlängerungsstudien untersucht. Dazu wurde DNA, die den 1,3-d(GpTpG) Cisplatin Trinukleotid-Schaden enthielt, hergestellt und mittels enzymatischen Verdaus charakterisiert. Mit dieser Methode konnte die Anwesenheit unerwünschter Nebenprodukte ausgeschlossen und die Entstehung des 1,3 Adduktes eindeutig nachgewiesen werden. Im Gegensatz zum 1,2-d(GpG) Addukt konnte Pol η alleine nicht über den 1,3-d(GpTpG) Cisplatin Schaden replizieren. Einzelnukleotid-Insertionsstudien zeigten, dass gegenüber dem 3’dG fehlerfrei ein dC eingebaut wurde, während der nächste Insertionsschritt nur mit erhöhten Enzymkonzentrationen erfolgte und mutagen war. Der Mechanismus des Replikationsblockes durch den 1,3-d(GpTpG) Cisplatin Schaden wurde mittels Röntgenstrukturanalyse auf atomarer Ebene untersucht. Dazu wurde Cisplatin-geschädigte DNA als Templat in Komplex mit dem katalytischen Fragment von Pol η, einem dATP in der aktiven Tasche und einem Primer mit terminaler 2’,3’-Didesoxyribose im zweiten Schritt der Verlängerung kristallisiert. Die Kristalle beugten die Röntgenstrahlung bis zu einer Auflösung von 2.5 Å. Im Kristall liegt der Komplex in zwei konformationell verschiedenen Formen vor. In beiden Formen (Komplex A und Komplex B) ist eine perfekte Watson-Crick Basenpaarung des 3’dGs (Pt-GTG) mit dem Primer zu erkennen. Dies erklärt die fehlerfreie Replikation des 3’dGs durch Pol η, welche sowohl in Zellen als auch in vitro beobachtet wurde. Das Hauptmerkmal der beschriebenen Struktur ist das zentrale Thymin des Schadens. Es befindet sich nicht zwischen den beiden Guaninen, sondern ist komplett aus dem DNA-Doppelstrang herausgedreht. Folglich ist das zentrale Thymin unfähig, den zweiten Verlängerungsschritt zu dirigieren, der nun gegenüberliegend zum 5’dG (Pt-GTG) erfolgt. Die Struktur beschreibt, wie der 1,3-d(GpTpG) Cisplatin Schaden eine Verlängerung durch Pol η verhindert. Das zentrale Thymin, das durch die Koordination des Platinatoms aus den Trinukleotidschaden herausgedreht ist, ist direkt vor dem Protein positioniert. Es tritt dadurch in sterische Wechselwirkung mit Methionin 74 und verhindert eine weitere Bewegung der Polymerase entlang des DNA-Strangs. Zudem ist zu sehen, warum die Inkorporation gegenüber dem 5’dG des Schadens so ineffizient verläuft. Die Rotation der DNA in die aktive Tasche des Enzyms (Komplex A → Komplex B) wird durch die Ausbildung einer Wasserstoffbrückenbindung zwischen dem dATP in der aktiven Tasche und dem 5’dG des Schadens getrieben. Dabei vergrößert sich aber der Abstand zwischen dem α-Phosphat des dATPs und der „gemodelten“ 3’OH-Gruppe des Primers. Der Abstand von ~8.5 Å ist für einen effektiven Nukleotidyltransfer eindeutig zu groß.

Vincent, Alan, and Rich celebrate the 100th episode of TWiV by talking about viruses with Nobel Laureate David Baltimore.

HIV-1 reverse transcriptase (RT) polymerase domain is known as target of nonnucleoside (NNRTIs) and nucleoside (NRTIs) reverse transcriptase inhibitors. However, resistance mutations in the polymerase domain lead repeatedly to therapeutic failure. The Connection (CN) and RNAse H domains of HIV-1 RT have recently gained more interest. They also might serve as independent new drug targets. Recently, in several in vitro studies several mutations in the CN and RNAse H domains were suggested to interfer with Polymerase resistance mutations (TAMs or finger domain mutations). In our in vivo approach, a clear differentiation between different HIV-1 subtypes was performed. Sequences of 57 HIV-1 subtype B infected patients were analysed. Their mutation status in the Connection and RNAse H domains was compared to a subtype-specific reference sequence. The sequences were studied for mutations. A potential correlation to Polymerase domain resistance mutation was analysed. Highly conserved amino acids and a number of natural polymorphisms were found for most of the studied positions. Subtype specific amino acid patterns were found. However, for positions 333, 359, 371, 390 and 558 a significant correlation to Polymerase domain resistance mutations was found. In conclusion, five positions were detected that might be involved in resistance mechanisms. The creation of a subtype specific reference sequence was necessary in order to distinguish between drug related mutations, subtype specific conservation or natural polymorphism. Extended sequence analysis to these regions are not recommended due to the small number of mutations found. Subtype speficication is highly recommended in order to gain resilient data.

On episode #62 of the podcast This Week in Virology, Vincent, Dickson, and Alan discuss STEP HIV-1 vaccine failure caused by the adenovirus vector, presence of West Nile virus in kidneys for years after initial infection, adaptation of the influenza viral RNA polymerase for replication in human cells, and the significance of the D225G change in the influenza HA protein. Host links Vincent Racaniello, Dickson Despommier, and Alan Dove Links for this episode: HIV vaccine failure probably caused by adenovirus vector used Persistence of West Nile virus in kidneys for years (JID and ProMedMail) (thanks, Lenn!) Adaptive strategies of influenza RNA polymerase for replication in humans New CDC estimates of 2009 H1N1 infection in US Receptor binding specificity of 2009 H1N1 virus Distribution of sialic acids in human respiratory tract

Vincent and Dickson continue Virology 101 with a discussion of how RNA viruses produce mRNA and replicate their genomes. Host links Vincent Racaniello and Dickson Despommier Links for this episode: Diagrams of viral RNA synthesis Animations of influenza virus and HIV-1 replication

Die vermehrte Anwendung antimikrobieller Wirkstoffe in der Human- und Veterinärmedizin hat zu einer Zunahme und Selektion antibakteriell resistenter Zoonoseerreger, wie Yersinia enterocolitica geführt. Resistenzmechanismen dieser Bakterien beruhen auf der Bildung chromosomal-kodierter ß-Laktamasen (A und B). Aufgrund unterschiedlichen Vorkommens der ß-Laktamase-Gene und der Expression dieser Enzyme innerhalb der einzelnen Y. enterocolitica Biotypen weisen diese eine heterogene Antibiotikaempfindlichkeit auf. In Deutschland stehen besonders Infektionen mit Y. enterocolitica Bioserotyp 4/O:3 an erster Stelle. Obwohl dieser Bioserotyp beide chromosomalen ß-Laktamasen bildet, wurden bei Y. enterocolitica Stämmen aus Bayern eine Amoxicillin/Clavulansäure-Antibiotika-Empfindlichkeit belegt. Ziel dieser Studie war das Vorkommen der Gene und die Expression für ß-Laktamase A und B bei 200 aus Bayern isolierten Y. enterocolitica 4/O:3 Stämmen mittels PCR und Agardiffusionstest zu untersuchen. Von den untersuchten Stämmen waren 60 humanen und 140 porcinen Ursprungs. Für den Nachweis der ß-Laktamase-Expression wurden im Doppelplättchenagardiffusionstest pro Stamm je zwei Müller-Hinton-Agarplatten mit einer Bakteriendichte von 107 KbE/ml beimpft und zusammen mit den zwei Antibiotikatestplättchen Ticarcillin (75 μg) und Amoxicillin/Clavulansäure (2/1 μg) bebrütet. Anschließend wurde die Expression der ß-Laktamase A und B anhand der Hemmhofgrößen um Ticarcillin und Amoxicillin/Clavulansäure bestimmt. Zur Überprüfung einer temperaturabhängigen ß-Laktamase-Expression wurde der Doppelplättchenagardiffusionstest bei den Bebrütungstemperaturen 30°C und 25°C durchgeführt. Im Anschluss wurden dieselben Stämme mittels konventioneller PCRMethode auf das Vorliegen der ß-Laktamasegene blaA und blaB überprüft. Die Auswertung der PCR Ergebnisse zeigte, dass bei 199 (99,5 %) der 200 untersuchten Y. enterocolitica 4/O:3 Stämme das blaA-Gen vorlag. Von diesen 199 Stämmen zeigten 198 Stämme auch eine BlaA-Expression in der Agardiffusion. Nur ein aus Schweine-Tonsillen isolierter Stamm zeigte trotz blaA-Gen keine BlaA-Expression. Bei einem der 200 untersuchten Stämme konnte mit Hilfe der PCR kein blaA-Gen nachgewiesen werden. Trotzdem zeigte dieser aus Hackfleisch isolierte Stamm im Agardiffusionstest eine BlaA-Expression. Das blaB-Gen wurde bei allen untersuchten Y. enterocolitica 4/O:3 Stämmen nachgewiesen. Im Agardiffusionstest exprimierten 3 humane und 1 porciner Stamm keine BlaB. Der Vergleich der Bebrütungs-temperaturen 30°C und 25°C zeigte einen Unterschied in der HHR-Größe um Ticarcillin. Während bei 30°C die meisten Stämme einen HHR von 3 mm aufwiesen, hatten die meisten Stämme bei 25°C einen HHR von 2 mm. Die durchgeführte Studie zeigt, dass nahezu alle (199/200) der aus Bayern isolierten und untersuchten Y. enterocolitica 4/O:3 Stämme die chromosomalen Gene für blaA und blaB besitzen. Zusätzlich konnte gezeigt werden, dass fast alle dieser Stämme beide ß-Laktamasen auch exprimieren. Die in einer vorangegangenen Studie ermittelte Empfindlichkeit dieser Stämme gegen Amoxicillin/Clavulansäure (20/10 μg) ist einerseits auf den 10fach höher konzentrierten Wirkstoff, andererseits auf eine geringere Expressionsmenge der ß-Laktamase B zurückzuführen. Die Bebrütungstemperatur zeigte keinen Einfluss auf die eigentliche ß-Laktamase-Expression, lässt aber einen Einfluss auf die Expressionsmenge vermuten.

Thu, 1 Jan 2009 12:00:00 +0100 http://jnnp.bmj.com/content/80/10/1181.short https://epub.ub.uni-muenchen.de/14987/1/A_variable_neurodegenerative_phenotype.pdf Meisel, A.; Zschenderlein, R.; Endres, M.; Siebert, E.; Lodi, T.; Baruffini, E.; Horvath, Rita; Prüss, H.; Stricker, S. ddc:610, Medizi

Seth Darst is a professor of Molecular Biophysics at the Rockefeller University in New York city, where his research centers on RNA polymerase, the enzyme at the heart of a cell’s ability to make protein from a set of DNA instructions. In this interview, I talk with Dr. Darst about how he got his start in research, whether computers will eventually be able to predict complex protein structures, and why eager young scientists shouldn’t miss their chance at postdoctoral training.

Eine akkurate DNA-Replikation ist notwendig, um die Stabilität der genetischen Information zu gewährleisten. Dieser Prozess wird durch DNA-Läsionen erschwert, die durch eine Vielzahl von Ursachen entstehen und häufig nicht vor dem Erreichen der S-Phase repariert werden können. Nicht nur kann durch Läsionen geschädigte DNA häufig nicht dupliziert werden, angehaltene Replikationsgabeln können auch zusammenbrechen und so zu DNA-Strangbrüchen führen. Die Funktion des RAD6-pathways liegt darin, die Umgehung (Bypass) von DNA-Läsionen während der Replikation zu ermöglichen, wodurch eine Toleranz gegenüber Schädigungen der DNA erreicht wird. In dieser Arbeit wurde die Regulation des RAD6-vermittelten Bypass von DNA-Läsionen durch posttranslationale Ubiquitin- und SUMO-Modifikationen des Replikationsfaktors PCNA untersucht. PCNA bildet einen trimeren Ring um die DNA und verstärkt durch Bindung der replikativen Polymerase deren Assoziation zur DNA und somit die Prozessivität der Replikation. Als DNA gebundener Faktor des Replikations-komplexes ohne katalytische Aktivität ist PCNA ideal geeignet, um durch seine Modifikation Replikations-assoziierte Prozesse zu regulieren. Die Ubiquitinierung von PCNA durch Enzyme des RAD6-pathways erfolgt als spezifische Antwort auf DNA-Läsionen während der Replikation und ermöglicht deren Bypass. Dabei bewirken unterschiedliche Ubiquitin-Modifikationen verschiedene Arten des Bypass. Die Mono-Ubiquitin-Modifikation führt zum Einsatz von speziellen Transläsions-Polymerasen, die eine größere Toleranz für geschädigte DNA haben, aber auch für die Entstehung von Mutationen verantwortlich sind. Einen mechanistisch anderen Bypass von DNA-Schäden bewirkt die Modifikation von PCNA mit einer Lysin K63-verknüpften Multi-Ubiquitinkette. Für diesen wird wahrscheinlich der neureplizierte, unbeschädigte Schwester-Strang als Vorlage benutzt. Unabhängig von Schädigungen der DNA wird PCNA während der S-Phase zusätzlich mit dem ubiquitin-ähnlichen Protein SUMO modifiziert. Dies führt zu einer Interaktion mit der Helikase Srs2, die als Antagonist zu dem zentralen Rekombinationsprotein Rad51 wirkt. Dadurch wird spezfisch die homologe Rekombination zwischen Schwesterchromatiden an der Rekombinationsgabel inhibiert, nicht jedoch andere Rekombinationsereignisse, wie. z.B. Rekom-bination zwischen homologen Chromosomen. Deshalb ist es wahrscheinlich, dass spezifisch die Replikationsgabel durch PCNA-SUMO-Srs2 geschützt wird, um schädliche Rekombination oder Rekombinationsstrukturen zu vermeiden, die mit Replikations-assoziierten Prozessen interferieren. Ubiquitin- und SUMO-Modifikation regulieren demnach unabhängige Prozesse. Interessanterweise haben diese aber eine verwandte Funktion im Bypass von DNA-Läsionen während der Replikation. Die Inhibition der Schwesterchromatid-Rekombination durch PCNA-SUMO-Srs2 lenkt den Bypass von DNA-Läsionen in einen durch PCNA-Ubiquitinierung gesteuerten Mechanismus.

We describe the isolation and characterization of an iridescent virus from commercially produced colonies of Gryllus bimaculatus in Germany, which showed apparent mortality. Transmission electron microscopy studies on adult cricket specimens revealed the paracrystalline assembly of icosahedral virus particles in the cytoplasm of hypertrophied abdominal fat body cells. The infecting agent could be cultivated in the lepidopteran cell line sf-9, where it caused cytopathogenic effects such as cell hypertrophy, cytoplasmic vacuolization, and cell death within 8 days postinfection. Negatively stained virus particles (n = 100) had dimensions of 172 +/- 6 nm (apex to apex) and 148 +/- 5nm (side to side). SDS-polyacrylamide gel electrophoresis of virus proteins showed more than 20 distinct polypeptides with a major species of approximately 50 kDa. Analysis of the restriction fragment length profiles from digestion of purified viral DNA with the endonucleases EcoRI, BamHI, and HindIII showed marked differences from the profiles of iridoviruses of lower vertebrates (genus Ranavirus), e.g., Rana esculenta Iridovirus and Frog virus 3. Restriction enzyme digests with the endonucleases MspI and HpaII indicated the lack of methylation of viral DNA. Polymerase chain reaction led to the amplification of a 420-bp gene fragment with 97 % sequence homology to the major capsid protein gene of Invertebrate iridescent virus 6 (IIV-6). The data indicate, that this new isolate, which is the first iridescent virus reported from Germany, belongs to the genus Iridovirus of the family Iridoviridae. It is proposed to be termed Gryllus bimaculatus iridescent virus (GbIV). The investigation of four other iridescent-like viruses which were obtained in 1998, 1999 and 2000 from various tissues of three reptile species, e.g. Pogona vitticeps, Chamaeleo quadricornis, and Chamydosaurus kingii, revealed cytoplasmic desoxyriboviruses with the exact morphological and molecular biological characteristics of the formerly described GbIV. We conclude that all five isolates of iridescent-like viruses from Germany are identical and represent variants or strains of the type species of the genus Iridovirus, IIV-6. The data indicate, that GbIV, an invertebrate iridescent virus, is able to replicate in reptiles.

Caliciviren sind kleine, unbehüllte Viren mit einem RNA-Genom positiver Polarität. Die Vertreter der Familie Caliciviridae sind Erreger wichtiger, zum Teil sehr unterschiedlicher Erkrankungen bei Mensch und Tier. Das Feline Calicivirus (FCV) gilt als einer der Haupterreger des Katzenschnupfenkomplexes. Das FCV ist für Replikationsstudien an Caliciviren besonders gut geeignet, da es sich effizient in Zellkultursystemen vermehrt. Da der Replikationszyklus nur zum Teil aufgeklärt ist, wurden in der vorliegenden Arbeit zwei Nichtstrukturproteine des FCV, das Vpg und die RNA-abhängige RNA-Polymerase (3D-Polymerase), näher charakterisiert. Die 3D-Polymerase ist für die Replikation der viralen RNA zuständig. Das Vpg ist ein viruskodiertes Protein, welches kovalent an den 5'-Terminus der genomischen und subgenomischen RNA gebunden ist. Gegen beide Proteine wurden zunächst erfolgreich polyklonale Antikörper generiert. Untersuchungen zur Expressionskinetik ergaben, dass die 3D-Polymerase in einer frühen Phase des Replikationszyklus exprimiert wird. Sie tritt auch beim FCV-Isolat KS20 nur in Verbindung mit der 3C-Protease als sogenanntes 3CD-Vorläuferprotein auf, freie Polymerase ist nicht nachweisbar. Im Gegensatz zur 3D-Polymerase liegt das Vpg-Protein in einer späteren Phase des Replikationszyklus des FCV in freier Form vor. Es wurden für das FCV-Isolat KS20 zwei mögliche Vorläuferproteine von etwa 43 bis 45 kDa und von 25 kDa detektiert, deren Funktion unklar ist. Die 3D-Polymerase und das Vpg wurden nur im Zytopasma der Wirtszelle lokalisiert. Beide induzieren keine neutralisierenden Antikörper. Ferner beschäftigte sich diese Arbei mit der Hypothese, dass das Vpg bei der Transkription der viralen RNA eine Rolle spielt und möglicherweise als Primer für die 3D-Polymerase dient. Zum anderen wurde untersucht, ob das Vpg an der Enkapsidierung der genomischen und subgenomischen RNA in die viralen Partikel beteiligt ist. Zum Nachweis potentieller Wechselwirkungen zwischen den einzelnen Proteinen wurde eine Co-Immunopräzipitation durchgeführt. Bei keinem der untersuchten Proteinpaare konnte eine Interaktion festgestellt werden.

Tue, 1 Jan 1991 12:00:00 +0100 https://epub.ub.uni-muenchen.de/8664/1/an_l_polymerase_deficient_rabies_virus_defective_interfering_particle_rna_8664.pdf Thiel, Heinz-Jürgen; Cox, James H.; Conzelmann, Karl-Klaus

Thu, 1 Jan 1981 12:00:00 +0100 https://epub.ub.uni-muenchen.de/5374/1/Zimmermann_Wolfgang_5374.pdf Zimmermann, Wolfgang

Tue, 1 Jan 1980 12:00:00 +0100 https://epub.ub.uni-muenchen.de/5377/1/Zimmermann_Wolfgang_5377.pdf Weissbach, Arthur; Bolden, Arthur; Chen, Shih Min; Zimmermann, Wolfgang ddc:610, Medizin